Download Application of Molecular Techniques to Improved Detection of

Application of Molecular
Techniques to Improved
Detection of Insecticide
Resistance
Kathleen K. Brewer
T
he significance and extent of resistance to insect and acarid control agents
is generally appreciated as a major obstacle to pest control, as well as an
environmental concern. Insecticide resistance is in transition from examination of gene products to the genes and their expression. Several gross chromosomal alterations already have been detected. Further details of these, as well as
the molecular basis of more subtle adaptations, can be expected to be revealed
shortly and validated in field-selected, economically significant pests.
There is precedence for this optimism. Localization and elucidation of nucleotide
sequences have been quickly followed by the development of DNA assays for TaySachs disease, hemoglobinopathies, and Lesch-Nyhan syndrome, to name only a
few. The human anomalies cited above are well-characterized biochemically and
represent messenger-RNA-abundant proteins, like many instances of metabolic
insecticide resistance. The availability of antibodies and related sequence data from
other species will help with the initial obstacle, which is localization of resistance
genes. The ability to perform crossing experiments with insects, as well as short
generation times and favorable polytene chromosomes in dipteran pests, also is
beneficial. Technical advances also suggest that many resistance mutations may be
revealed shortly. For example, newer vectors and techniques to sequence DNA
directly have accelerated mutation research by bypassing tedious, and more risky,
cloning steps in cases where the wild-type sequence is known. These observations,
as well as the recent explosion of interest in this area, suggest that it is not premature
to think about ways in which investigations of the molecular basis of insecticide
resistance might contribute to pest management.
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AMERICAN ENTOMOLOGIST
Various methods are available to estimate the frequency of resistant phenotypes.
Field trials, dose response, and discriminating dose bioassays initially are useful for
documenting the presence of adaptation or actual resistance and for estimating the
efficacy of control agents. Several authors have indicated that more specific and
sensitive methods are needed for estimating the frequency of resistance mutations
in both pests and beneficial insects before control failures occur, as well as for
evaluation of management tactics. A related topic, plant-insect interactions and
host-race formation, also would gain from more sensitive means to detect genetic
heterogeneity. Molecular techniques already have been applied to herbicide, antibiotic, and antineoplastic resistance, and these applications could provide useful
paradigms for entomology. Here, requirements for resistance assays are summarized, gaps in detection of insecticide resistance identified, followed by discussion
of potentially useful molecular tools. These techniques undoubtedly
are wellknown to molecular biologists, but may be unfamiliar to other entomologists.
Here, the emphasis is on resistance to synthetic insecticides. However, the natural
variation in sensitivity and evolution of resistance to Bacillus thuringiensis in field
and artificial situations, for example, suggest that a means to detect genetic heterogeneity to this and other biopesticides also is needed.
he ideal assay for detecting resistance is sensitive enough to test single insects,
specific for a resistance factor, and capable of resolving heterozygous and
homozygous genotypes. The assay should be adaptable to screening large
numbers of insects and should be able to provide information regarding the selectivity of control treatments. The assay also should identify a discriminating dose,
and the system should permit more than one test per specimen. The ultimate goal
includes the development of cost-effective and widely accessible assays that can be
implemented in the field. In addition, a means to simultaneously detect the presence
of pathogens is desirable for disease vectors. Assays with many of these features
have been developed for esterases, glutathione-S-transferases
(GSTs), and resistant
forms of acetylcholinesterase.
T
Requirements
Microassays
T
Gaps in Detection
Resistance
here are instances where microassays for metabolic resistant factors have
not been forthcoming or are incomplete. (The following summary provides
little in the way of new information or syntheses but I've included it here
to put the potential of molecular tools in perspective.) The development of single
insect assays for the mixed function oxidases (MFOs) has been complicated by
problems with extraction, stability, purification, and overlapping substrate preferences of isoenzymic forms. The GSTs also have a pattern of overlapping catalytic
capability without real specificity. Measurement of general esterase activity with a
surrogate substrate may not distinguish between variants with enhanced titers versus
mutants with altered specific activity. Assays that test whole enzyme titers can be
misleading, because only one isozyme actually may be involved in resistance. Also,
there are cases of metabolic resistance where additional information regarding the
genotype is essential. For example, Devonshire and his colleagues have combined
an immunoassay with a DNA probe to identify resistant genotypes in Myzus
persicae (Sulzer). This has practical consequences because revertants constitute
obscured resistance potential within the population. There is a parallel to this in
prenatal diagnosis of a lipid storage disorder, in which the enzyme assay cannot
resolve intractable infantile and less severe adult phenotypes; molecular analysis is
required to make this distinction.
There are also adaptations of the nerve insensitivity type that may require DNA
probes. Alterations in the expression of sodium channel protein and the phospholipid content of membranes have been correlated with nerve insensitivity. These
might be assayed with a quantitative immunological assay or biochemically, respectively. But nerve insensitivity also is believed to include structural mutations
in target macromolecules-the
sodium channel and gamma-aminobutyric-acidchloride ionophore complex. Such structural mutations could be refractory to
Summer 1991
for Resistance
of Insecticide
97
immunological assays because they are likely to be
which may be lost with solubilization. So there are
been refractory to the development of microassays,
from assays directed toward the gene, or in addition
Molecular Techniques with the
Potential for Application to Detection
of Insecticide Resistance
changes in the configuration,
cases of resistance that have
and others that might benefit
to the gene product.
T
here are both indirect and direct methods for detection of mutations that
might have application to integrated pest-management (IPM). (The descriptions provided here are intended for orientation only.) Linkage analysis is
an indirect tool that relies on markers coinherited with a mutation at a statistically
reliable frequency. Markers can be phenotypic traits such as eye color, but DNA
markers, known as restriction fragment length polymorph isms (RFLPs),are used
more often. The molecular basis of RFLPs are benign single-base alterations scattered throughout the population. These polymorphisms are sensitive to digestion
with restriction enzymes (fig. 1). Restriction enzymes are bacterial-derived endonucleases that usually recognize and cleave (A) palindromic sequences-that
is, shorr
stretches of nucleotides that read the same in both directions, as in the example
below for Hpa II.
(5')CC-G-G(3')
(3')G-G-CC(5')
Patterns of restriction fragments are visualized by electrophoresis (fig. 2). Linkage
analysis can be invaluable for screening when the gene has not been isolated, as in
Huntington's disease, or when many mutations cause a similar phenotype.
Fig. 1. Two means of detecting
mutations. Restriction fragment
analysis (above). Nucleotide changes
can be associated with restriction
enzyme site cleavage. This is useful
when the change is benign
(polymorphism) but is coinherited
with a deleterious phenotype.
Restriction fragment analysis also can
be used when a mutation itself
changes the pattern of restriction
enzyme digestion. Alternatively,
(below) mutations can be directly
detected by hybridizing with a
nucleotide probe to the normal
sequence. Because the mutation causes
a mismatch, it is unstable and will be
washed off, in contrast to normal.
Restriction fragment analysis
~-_•... ~-.-----~
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Allele-specific oligonucleotide hybridization
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98
Target nucleic acid
Hybridization probe
Point mutation
Site of enzyme action
AMERICAN ENTOMOLOGIST
~
CLEAVE
Fig. 2. Traditional means of
detecting RFLPs. Total (genomic)
DNA is cut with restriction enzymes
and separated by electrophoresis.
DNA is then transferred to an
immobilizing filter. A probe with some
type of intensifying signal is added.
The unbound probe is washed off
following an incubation period.
Patterns of RFLPs are revealed
following autoradiography.
The
polymerase chain reaction can
eliminate the need for the
hybridization step, since amplified
and digested fragments are visible
following electrophoresis.
DNA
~---
WITH RESTRICTION
-\
AGAROSE
ENZYMES
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GEL ELECTROPHORESIS
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NITROCELLULOSE
GEL
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1/1
HYBRIDIZE WITH
LABELED PROBE
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WASH
1/2
2/2
Alleles (kb)
~3.0
L=.J
2.0
RFLPs
Probe
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Allele 1
3.0 kb
Allele 2
Alternatively, mutations may be revealed directly by hybridization studies, the
essence of which is the difference in binding stability between a chain of nucleotides,
referred to as a probe, containing the wild-type sequence and a corresponding
stretch of mismatched DNA or RNA (fig. 1). Samples of DNA bound to a solid
support are incubated with a probe that corresponds to the sequence of interest.
When washed under conditions of correct stringency, only probes that match
perfectly remain. Two conditions must be met for this to work. First, the probe
must find its target sequence. A larger nucleotide probe can find its complementary
sequence in the vast amount of DNA that constitutes a genome more easily than
a smaller probe, or when nature has provided an increased copy number of the
target sequence. Second, washing conditions must be such that only perfectly
matched probes remain. As a general statement, gross chromosome rearrangements,
deletions, or insertions are detected most readily as described here. Gene amplification has been shown to be the molecular basis of two cases of resistance to
date, and quantitative hybridization assays are available for these.
Many as yet undiscovered resistance mutations are believed to be single nucleotide substitutions, and these subtle adaptations require more sensitive means to
Summcr
I f.)f.) I
99
detect them. Formerly, probes of at least 100 nucleotides in length were required
to find their corresponding sequences in total (genomic) DNA. But probes of this
size have small differences in thermal stability between a perfectly matched hybrid
and one containing a single mismatched base pair. Consequently, single nucleotide
substitutions usually could not be detected with probes of this size unless they
fortuitously abolished or created a restriction enzyme site, as in sickle-cell anemia.
But palindromic sequences are represented by approximately one in every six nuc1eotides. For example, only about half of human disease mutations are detectable
by this method, illustrating the considerable limitation to this approach. The laborintensive, multistep, and capricious nature of conventional hybridization analysis,
as well as slow turnover time, limited applications of this technique. Within· the
past few years, polymerase chain reaction (PCR) technology has provided a means
to increase the copy number of any targeted sequence. This has increased the
sensitivity and accessibility of hybridization studies in general. Detection of point
mutations is much easier following PCR. Shorter probes (seventeen to twenty-one
nucleotides) can find their targets more readily, since there are a billionfold copies.
In addition, shorter probes are more sensitive because they are readily washed off
if a single mismatch (mutation) is present (fig. 1).
The PCR is an in vitro amplification scheme consisting of a series of incubation
steps at various temperatures, starting with extremely small amounts of DNA for
the initial substrate. Each cycle consists of the following steps (fig. 3):
1. DNA is heat denatured to single-stranded templates.
2. Flanking nucleotide primers are annealed to their corresponding sequences at
a low temperature. These primers are short nucleotide chains that do just what
the term implies-they
are obligate starting points for the polymerase enzyme.
3. The thermostable
polymerase fills in corresponding
nucleotides at a higher
temperature, using the single-stranded DNA as a template and four nucleotide
building blocks.
Thus, each strand is reproduced with each cycle and is a template for the subsequent
reaction, resulting in an exponential increase in copy number of a desired nucleotide
sequence within hours. The results of this reaction can be easily visualized by
electrophoresis in the presence of ethidium bromide, a dye that is selective for
nucleic acids.
Several applications of the PCR have potential for improved detection of insecticide resistance. RFLPs or mutations that cause the loss or gain of a restriction
site may be studied more easily with this method because amplified and digested
DNA can be visualized without hybridization. When hybridization is required,
nucleotide probes to normal and mutant sequences are selected. They are long
enough to represent unique sequences in the genome, yet short enough to be
destabilized by a single internal mismatch.
There are instances where metabolic resistance factors might be studied in messenger RNA (mRNA), for which the PCR also is applicable. This is done by making
complementary DNA (cDNA) from an RNA template using reverse transcriptase.
This enzyme (reverse transcriptase) is so called because it works in the opposite
direction from normal transcription, making DNA from RNA. cDNA is that portion
of a gene that an organism actually uses; in other words, noncoding, extra genetic
material is not included. In a second reaction, PCR is performed as already described. This two-step process is less complicated than it sounds. mRNA does not
have to be isolated from other RNA species, and both reactions can be done in
the same buffer system. Metabolic resistance variants with more enzyme protein
versus mutants with more active enzyme species might be resolved in this manner.
This approach has been used to screen for thymidylate synthase in cisplatin-resistant
tumors. It might be necessary also to look at mRNA when the proteins of interest
are difficult to extract from single insects, as for the MFOs.
Additional applications of the PCR also have potential for IPM. Because this
technique permits detection of minute samples of DNA, infectious agents-those
of public health interest, as well as plant pathogens-can
be detected in pest
populations, as has been shown for the Lyme disease spirochete. The tools of
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AMERICAN ENTOMOLOGIST
original
DNA
peR primer
new DNA
DNA + primers + dNTPs
+ DNA polymerase
+
••I
denature and synthesize
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Fig. 3. Polymerase chain reaction.
DNA is separated (denatured) by
heating. Probes specific for the target
sequence prime the synthesis of new
DNA in the presence of nucleotide
building blocks (dNTPs) and DNA
polymerase, a DNA replicating
enzyme. Because each new strand of
DNA is a template for the subsequent
reaction, sequential cycles cause
exponential amplification, resulting in
greater than a millionfold increase of
a target fragment more than twenty to
forty cycles.
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continue for 20-40 cycles
linkage analysis and hybridization are hardly new, but more recent techniques have
made them potentially useful tools for IPM.
T
he nucleic acid approaches outlined above are specific and sensitive enough
to assay single, even minute, insects. Direct detection using either probes
or restriction enzyme digestion has excellent resolution because wild-type,
heterozygous, and homozygous genotypes can be distinguished. Detoxifying enzymes can vary with developmental stage and diet-a possible variable in sampling
for metabolic studies-but not for DNA assays. It appears likely that multiplex
screening of each specimen, either metabolic, immunological, or genetic, would
be compatible with DNA diagnostics. Simultaneous detection of pathogenic organisms in disease vectors and monitoring of insect pathogens and parasites, either
by immunological or molecular means, seems feasible too. Also on the positive
side, the peR is a well-established technique, which is accessible to investigators
without previous experience in molecular biology. The reaction is relatively robust,
once conditions are optimized. Preliminary experiments generally involve finding
the most efficient sets of primers and determination of the optimal annealing
Summer 1991
Anticipated Advantages and
Limitations to Detection of Insecticide
Resistance Using Molecular
Techniques
101
temperature. However, there will be biological, statistical, and technical obstacles
and limitations to the application of DNA diagnostics to IPM.
The major caveat in interpretation of DNA diagnostics is that they test only the
genotype, unlike the best assays, which include the feature of testing response to
control agents. Primary discriminating dose assays provide response data. In addition, DNA diagnostics are limited by their specificity; more than one mutation
might confer the same resistant phenotype. However, most cases of resistance are
believed to be caused by a single mutation within a population. Sessile and parthenogenetic insects could be notable exceptions, because they actually may be
series of isolated demes within populations. Estimations of polygenic resistance
may be inflated by studies with laboratory-selected
insects, because there are good
reasons to believe that such experiments tend to select for multifactorial resistance.
Linkage analysis may prove to be somewhat dynamic with respect to specificity,
because the coinheritance of genes can drift within a population over time. Note
also that restriction enzymes may fail to cut if any of the nucleotides in the
recognition sequence or cleavage site are altered; consequently, analysis by this
method is slightly less precise. Limitations because of specificity may vary with the
biology of the pest, the selection conditions, and the assay.
Insecticide resistance is essentially a population phenomenon.
This is a crucial
point in adapting DNA diagnostics to IPM. It is believed that several hundred
insects are requjred to detect resistance mutations before control failures occur.
But the number of samples required for molecular assays will be a subset of the
initial bioassay. DNA or RNA sample preparation will be dictated by the application.
Sometimes cell lysates are sufficient preparation for molecular studies. Merely
squashing insects on a nylon filter can be adequate for hybridization studies if
multiple copies of the target sequence are already present. The PCR requires varying
degrees of nucleic acid purification. Because of the amplification potential of this
technique, only a small amount of starting DNA is required, so inhibitors are
diluted. In addition, the initial heat denaturation step eliminates many other troublesome agents. Simple cell lysates and a nuclease inhibitor often suffice. This is
not the case when the target is an infectious agent present in only a small proportion
of cells, or when the PCR is inefficient, as paradoxically occurs for some sets of
primers. In these instances, more target DNA is needed and therefore, more rigorous
purification is indicated. Restriction enzymes also are sensitive to contaminants and
indeed may have anomalous activity in their presence.
Complete DNA or RNA purification involves lysis of cells, inhibition of endogenous nucleases, degradation of membrane components, and pelleting of the nuclear fraction. Because mRNA is in the cytoplasm, this fraction is saved for RNA
extraction. Proteins are removed by phenol-chloroform
extractions, followed by
selective ethanol-salt precipitation of nucleic acids. Because of the tissue-specific
nature of many mRNAs that are relevant to resistance and the ubiquitous and
hardy nature of endogenous RNA-degrading enzymes, purified RNA often may be
required. These RNA protocols probably will have to be individualized, because
methods to isolate RNA do not work equally well with tissues from various sources.
Sample preparation for molecular assays, then, will vary from simple to rigorous.
While the power of the PCR is partially because of its amplification potential,
this also raises some unique considerations for preparation of DNA and RNA from
insects. Fertilized gametes will have to be excluded or accounted for. Homologous
or partially homologous sequences in microbial symbionts also could confound
quantitative studies or introduce PCR artifacts. Experimental controls for cross
contamination between samples and reagents always are necessary.
The PCR reaction and subsequent steps are not particularly labor intensive. A
single worker can process approximately 120 samples per day using an automated
thermal cycler. A mini-electrophoresis
apparatus, similar to that used for screening
monoclonal antibodies, is useful for examination of many peR products. Dot blot
formats allow hybridization of many samples simultaneously.
When probes are
required, a means to visualize them after hybridization is necessary. This is done
by replacing the terminal phosphate of short probes with some type of reporter
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AMERICAN ENTOMOLOGIST
molecule, usually a radioactive isotope. But chromogenic nonradioactive reporter
schemes that are permanent and less hazardous also are available. Signal intensity
with both of these types of reporters usually is enough to permit overnight development. The greatest bottleneck in application of molecular techniques to detection
of insecticide resistance is likely to be the amount of sample preparation required.
There are universal technical considerations as well. The nucleotide sequence
of at least one single copy region adjacent to, or including, the mutation or RFLP
must be available so that complementary
nucleotide primers can be synthesized.
The size of the genome must be estimated for each pest so that primers and probes
that are likely to hybridize to unique sequences can be designed.
Also on the debit side, it is impossible to imagine how nucleic acid assays could
be conducted in the field. But this limitation applies to some of the metabolic and
immunological assays as well, and it does not preclude their use. While there are
expected benefits from the application of molecular techniques to rPM, there also
will be limitations and practical problems to be resolved.
ome molecular assays can be expected to have more widespread application
than others. Metabolic resistance adaptations may be either quantitative or
qualitative. All manner of favorable mutations seem possible. Deleterious
mutations in the well-studied human beta-globin gene, for example, provide a
reverse analogy for how heterogeneous the molecular basis of metabolic resistance
might be. As previously mentioned, the number of resistance mutations within
interbreeding populations is thought to be limited but could be variable between
reproductively isolated populations. Linkage analysis can be most useful in situations like this when it is not possible or not cost-effective to screen for every
mutation.
Some nerve insensitivity adaptations are likely to be structural mutations, and
these are expected to be relatively homogeneous at the molecular level because of
requirements for normal signal transmission by the target macromolecules.
Such
nerve insensitivity factors may be the best candidates for direct assays because a
more limited battery of nucleotide probes would be required.
Nucleic acid assays will not supplant other procedures. The limitations of diagnosis by genotype indicate that this approach should be confined to cases of
resistance that do not lend themselves to assays for gene products or that would
benefit from a dual phenotypic and genotypic assay. Indirect tools such as linkage
analysis will be most useful initially; some will be replaced later by direct tests.
Where insect collection or extensive sample preparation is impractical, molecular
assays may have utility only in laboratory simulation studies.
Less is known about variation in susceptibility to biological control agents and
genetically engineered crops. It is hoped that all available technology will be recruited for detection of genetic heterogeneity to these newer agents as well.
0
S
Expected Contribution
of Molecular
Techniques to Insecticide Resistance
Management
L. B. Brattsten, A. L. Devonshire, R. T. Roush, R. M. Sawicki, and D. M.
Soderlund generously provided reprints or galley proofs of manuscripts in press.
C. W. Holyoke, Jr., and anonymous reviewers made very helpful comments on
earlier versions of this manuscript.
Acknowledgment
Kathleen. Brewer received her masters of science degree in entomology from the
University of Delaware. She is completing predoctoral studies at Thomas jefferson
University. Her research problem deals with the molecular basis of a Tay-Sachs
disease variant.
Summer
1991
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